BCL-2 family protein interactions regulate apoptosis, an orchestrated set of events designed to rid the body of damaged or unwanted cells, and a process critical for normal development and organism homeostasis (Adams, J. M., and Cory, S. (1998), Science 281, 1322-1326; Danial, N. N., and Korsmeyer, S. J. (2004), Cell 116, 205-219). Whereas multidomain anti-apoptotic proteins such as BCL-2 guard against cell death, multidomain pro-apoptotic proteins such as BAX constitute a gateway to cell death through mitochondrial damage. Multidomain apoptotic proteins display sequence conservation in up to four BCL-2 homology (BH) domains, yet the “BH3-only” subset of pro-apoptotic proteins displays homology only to the third BH domain. The BH3-only proteins function as death sentinels situated throughout the cell, poised to transmit signals of cell injury to multidomain members. Depending on the nature of apoptotic stimuli and cellular context, the BH3-only protein's death signal will either be neutralized by anti-apoptotic proteins or delivered, directly or indirectly, to the mitochondrial executioners BAX and BAK. When activated, these pro-apoptotic multidomain members induce permeabilization of the outer mitochondrial membrane, enabling released mitochondrial factors to activate caspases, which irreversibly execute the death program (Green, D. R. (2005). Cell 121, 671-674).
Structural studies have demonstrated the insertion of BH3 ligands into the hydrophobic groove formed by the juxtaposition of BH1, BH2 and BH3 domains of anti-apoptotic multidomain members (Day, C. L., et al. (2005), J Biol Chem 280, 4738-4744; Denisov, A. Y., (2003), J Biol Chem 278, 21124-21128; Liu, X., et al. (2003), Immunity 19, 341-352). Recent work has uncovered the binding specificities of BH3-only members for their anti-apoptotic partners and the structural basis for these selectivities (Certo, M., Moore, et. Al. (2006), Cancer Cell 9, 351-365.; Chen, L., et al. (2005); Mol Cell 17, 393-403). BH3-only proteins antagonize anti-apoptotic proteins through these direct interactions, and thereby decreases anti-apoptotic inhibition of BAX/BAK and/or competitively displace bound BH3-only members that are capable of direct activation of BAX/BAK. BH3-only proteins that exclusively engage anti-apoptotic proteins to exert their pro-apoptotic activity (e.g. BAD) have thus been termed “sensitizers”(Letai, A., et al. (2002), Cancer Cell 2, 183-192) or “derepressors” (Kuwana, T., et al. (2005)1 Mol Cell 17, 525-535). Recent yeast two-hybrid, immunoprecipitation, and biochemical studies support a role for select BH3-only members, including BID and BIM, in direct activation of BAX. (Carton, P. F., et al. Mol Cell 16, 807-818 (2004); Harada, H., et al. PNAS USA 101, 15313-15317 (2004); Marani, M., et al. Mol Cell Biol 22, 3577-3589 (2002); Walensky, L. D., et al. Mol Cell 24, 199-210 (2006); Wang, K., et al. Genes Dev 10, 2859-2869. (1996). However, the inability to measure a direct binding affinity between the BH3 domain of these “activator” BH3-only proteins and native BAX protein, and the requirement of up to 50 micromolar dosing of activating BH3 peptides to trigger BAX activation in vitro (Kuwana, T., et al. (2005). Mol Cell 17, 525-535; Kuwana, T., et al. (2002). Cell 111, 331-342), have led to uncertainty about the existence of a BH3-mediated direct BAX/BAK activation pathway. In fact, the lack of binding and structural data to support such interactions has led to the hypothesis that the pro-apoptotic potency of presumed activator peptides, such as BIM BH3, may instead reflect their ability to effectively neutralize all anti-apoptotic proteins tested to date.